INACTIVATION OF SENSITIZATION IN SENSORY NEURONS

已知炎性物质如前列腺素（PG）使感觉神经元对随后的刺激敏感，导致称为痛觉过敏的敏感性提高的状况。目前我们认为炎症性PGS通过激活cAMP转导级联反应增强感觉神经元的敏感性和兴奋性。然而，很少有人知道有关的监管机制和转导级联控制的强度或持续时间的敏感性增强，导致神经方面的炎症反应和痛觉过敏。所提出的研究试图建立生理机制，从而钙/一氧化氮/cGMP途径导致感觉神经元的增强的兴奋性或敏化的失活。该提议的假设是cGMP通过激活cGMP依赖性蛋白激酶（PKG）及其对其他细胞内介质的可能调节，改变各种膜电流的性质或状态，以逆转由cAMP/PKA途径产生的增强的神经元兴奋性。本研究将使用培养的大鼠背根神经节细胞作为模型系统。这些神经元的电生理特性将被检查与膜片钳技术。这允许测量由整个细胞或单离子通道产生的膜电流。结合膜片钳记录，将测量细胞内钙、cAMP和cGMP浓度的变化，并将其与观察到的神经元兴奋性变化相关联。本研究的具体目的是：1）利用荧光钙指示剂定量测定细胞内钙离子浓度的变化，从而直接确定钙对敏化失活信号通路激活的贡献;（二）建立对不同转导级联的基本理解，并确定失活途径是否对刺激的性质具有特异性或更广义的细胞设计的一部分; 3）确定引起致敏失活的特异性细胞内介质及其作用机制。这将提供确定由失活介质调节的特定靶蛋白的初始步骤。最终，如果我们能够建立调节兴奋性的上调和下调的调节机制，就有可能设计出选择性调节改变的通路的疗法，从而抑制与慢性炎症性疾病（如类风湿性关节炎）相关的持续性疼痛和敏感性升高。

Inflammatory agents such as the prostaglandins (PGs) are known to sensitize sensory neurons to subsequent stimulation, resulting in a condition of heightened sensitivity known as hyperalgesia. At present we that inflammatory PGS enhance the sensitivity and excitability of sensory neurons through activation of the cAMP transduction cascade. However, very little is known regarding the regulatory mechanisms and transduction cascades controlling the intensity or duration of the enhanced sensitivity that results in neurogenic aspects of the inflammatory response and hyperalgesia. The proposed studies seek to establish the physiological mechanisms whereby the calcium,/nitric oxide/cGMP pathways lead to inactivation of the enhanced excitability or sensitization of sensory neurons. The hypothesis of this proposal is that cGMP, through activation of cGMP- dependent protein kinase (PKG) and its possible modulation of other intracellular mediators, alters the properties or state of various membrane currents to reverse the enhanced neuronal excitability produced by the cAMP/PKA pathway. The studies outlined in this proposal will use rat dorsal root ganglion cells grown in culture as a model system. The electrophysiological properties of these neurons will be examined with the patch-clamp technique. This allow measurement of membrane currents arising from the whole cell or single-ion channels. In conjunction with patch-clamp recordings, changes in the concentration of intracellular calcium, cAMP, and cGMP will be measured and correlated to the observed alterations in neuronal excitability. The specific aims of this proposal are: 1) To quantify the changes in intracellular calcium concentration using fluorescent calcium indicators and thus determine directly the contributions of calcium to the activation of signaling pathways involved in the inactivation of sensitization; 2) To establish a fundamental understanding of the different transduction cascades and to determine whether the inactivating pathways are specific to the nature of the stimulus or part of a more generalized cellular design; 3) To determine the specific intracellular mediators and their mechanisms of action that give rise to the inactivation of sensitization. This will provide an initial step in determining the specific target proteins that are modulated by the mediator(s) of inactivation. Ultimately, if we can establish the regulatory mechanism modulating the up and down regulation of excitability, it will be possible to design therapies that selectively modulate the altered pathway and thus curb the persistent pain and heightened sensitivity associated with chronic inflammatory conditions, such as rheumatoid arthritis.